Human pericytes for ischemic heart repair

Abstract

Human microvascular pericytes (CD146(+)/34(-)/45(-)/56(-)) contain multipotent precursors and repair/regenerate defective tissues, notably skeletal muscle. However, their ability to repair the ischemic heart remains unknown. We investigated the therapeutic potential of human pericytes, purified from skeletal muscle, for treating ischemic heart disease and mediating associated repair mechanisms in mice. Echocardiography revealed that pericyte transplantation attenuated left ventricular dilatation and significantly improved cardiac contractility, superior to CD56+ myogenic progenitor transplantation, in acutely infarcted mouse hearts. Pericyte treatment substantially reduced myocardial fibrosis and significantly diminished infiltration of host inflammatory cells at the infarct site. Hypoxic pericyte-conditioned medium suppressed murine fibroblast proliferation and inhibited macrophage proliferation in vitro. High expression by pericytes of immunoregulatory molecules, including interleukin-6, leukemia inhibitory factor, cyclooxygenase-2, and heme oxygenase-1, was sustained under hypoxia, except for monocyte chemotactic protein-1. Host angiogenesis was significantly increased. Pericytes supported microvascular structures in vivo and formed capillary-like networks with/without endothelial cells in three-dimensional cocultures. Under hypoxia, pericytes dramatically increased expression of vascular endothelial growth factor-A, platelet-derived growth factor-β, transforming growth factor-β1 and corresponding receptors while expression of basic fibroblast growth factor, hepatocyte growth factor, epidermal growth factor, and angiopoietin-1 was repressed. The capacity of pericytes to differentiate into and/or fuse with cardiac cells was revealed by green fluorescence protein labeling, although to a minor extent. In conclusion, intramyocardial transplantation of purified human pericytes promotes functional and structural recovery, attributable to multiple mechanisms involving paracrine effects and cellular interactions.

Figure 1. Survival rate and cardiac functional assessment

(A) Cumulative survival rate of the animals over 8 weeks post-surgery (Kaplan-Meier Survival Curve, log-rank test p=0.529). Echocardiographic analyses revealed a significant reduction of LV dilatation by transplantation of all three pericyte populations (AP, FP1, and FP2), as shown by the smaller LV area in end-diastole (B) and end-systole (C) of hearts at both time points. Injection of pericytes also resulted in substantial improvement in LV contractility, as indicated by greater fractional shortening (D), fractional area change (E), and ejection fraction (F), at both time points. (^†p≤0.001; ^§p≤0.005; ^#p≤0.01; ^*p≤0.05; versus PBS control group at each time point)

Figure 2. Attenuation of myocardial fibrosis by pericyte treatment

(A) Masson’s trichrome-stained transverse sections of hearts injected with pericytes or PBS (collagen in blue/purple, cardiac muscle in red; scale bars=1 mm). (B) The fibrotic area fraction was dramatically decreased in pericyte-injected hearts (p≤0.001). (C) Pericyte group had no significant increase in the infarct wall thickness. (D) When culturing with hypoxic pericyte-conditioned medium (P-CM), the proliferation of murine cardiac fibroblasts was significantly reduced (^†p=0.019, versus normoxic P-CM) while muscle fibroblast proliferation exhibited the same pattern (*p<0.001). Normoxic P-CM had a pro-proliferative effect over control medium in muscle fibroblasts, but not in cardiac fibroblasts (^#p<0.05). Skeletal myoblast proliferation was not significantly affected by either of the P-CMs. (E) Expression of MMP-2 in cultured pericytes was higher than that in skeletal muscle lysates. Conversely, MMP-9 expression in pericytes was nearly 10 times less (logarithmic scale of 10 in arbitrary fluorescence units). (F) Expression of both MMP-2 and -9 in pericytes did not change significantly under hypoxia (p>0.05, logarithmic scale of 10 in arbitrary fluorescence units). (G) Immunohistochemistry revealed MMP-2 expression (red arrows) by some of the GFP-labeled donor pericytes (green arrows) within the infarct area at 2 weeks post-infarction (a) merge (b) anti-GFP in green (c) MMP-2in red (d) DAPI nuclei staining in blue (scale bar=20μm).

Figure 3. Reduction of host phagocytic cell infiltration by pericyte transplantation

(A) H&E staining revealed a greater focal infiltration of leukocytes (dark blue-stained nuclei) within the infarct region in PBS-injected controls at 2 weeks post-infarction (scale bars=100μm). (B) Anti-mouse CD68 immunostaining showed that the infarct region of pericyte-injected hearts contains less host phagocytic cells (scale bars=50μm). (C) Host CD68-positive cells were locally attracted to the infarct region but not to the unaffected myocardium (posterior ventricular wall) in both groups (scale bars=50μm). (D) Host monocytes/macrophage infiltration at the infarct site was significantly reduced (p<0.001). (E) The proliferation of murine macrophages was significantly inhibited when culturing with pericyte-conditioned media (*p=0.018, ^#p<0.001, versus control medium), an effect more prominent with hypoxic pericyte-conditioned medium (^†p=0.002, hypoxia versus normoxia). (F) Cultured pericytes exhibited sustained, high expression of genes regulating the inflammatory responses, even under 2.5% O₂ (N: normoxia; H: hypoxia). Little expression of IL-1α and no expression of IL-4, IL-10, iNOS, 2,3-IDO, TNF-α, and IFNγ were detected. (G) No statistically significant difference in expression of genes of immunoregulatory molecules between normoxic- and hypoxic-cultured pericytes except MCP-1, which notably decreased in hypoxic cultures (sqRT-PCR analysis, p=0.027).

Figure 4. Promotion of host angiogenesis by pericyte treatment

Representative images of anti-mouse CD31 immunostaining (A) in the peri-infarct area and (B) within the infarct region of hearts injected with pericytes or PBS (scale bars=50μm). (C) Pericyte-treated hearts displayed significantly higher capillary densities in the peri-infarct area (p<0.05) and within the infarct region (p<0.001).

Figure 5. Pericytes support microvascular structures

(A) While HUVECs seeded onto Matrigel-coated wells formed typical capillary-like networks after 24 hours, (B) pericytes formed similar structures within 6–12 hours (scale bars=1mm). (C) When co-cultured on Matrigel, dye-labeled pericytes (green) and HUVECs (red) co-formed capillary-like networks within 6–12 hours, ([C], inset) with collocations of pericytes and HUVECs in three-dimensional structures formed 24 hours after seeding (scale bars: main=200μm; inset=100μm). (D) HUVECs (red) appear to line and spread out on top of the pericyte-formed structures (green) (scale bar=100μm). (E) To simulate native capillary formation, HUVECs were evenly encapsulated into 3D Matrigel plug for 72 hours but unable to form any organized structure (scale bars=1mm). (F) Pericytes instead formed capillary-like networks in Matrigel plug with structural organization and maturation over time (scale bars=1mm). (G) When dye-labeled pericytes (green) and HUVECs (red) were co-casted into the 3D-gel plug, the two types of cells formed microvessel-like networks within 72 hours, (H) with pericytes surrounding HUVECs (scale bars: G=200μm; H=50μm).

Figure 6. Expression of pro-angiogenic factors and associated receptors under hypoxia

(A) Pericytes dramatically up-regulated VEGF-A, PDGF-β, TGF-β1 gene expression under hypoxic conditions (2.5% O₂) while expression of other pro-angiogenic factors, including bFGF, HGF and EGF were distinctively repressed. (B) Simultaneously, VEGFR-1 (Flt-1) and -2 (Flk-1) were substantially up-regulated, and PDGF-Rβ expression was moderately increased. All expression levels are normalized to human cyclophilin and presented in arbitrary fluorescence units on an expanded logarithmic scale (#p<0.05, *p≤0.001, †p<0.01, hypoxia versus normoxia). (C) Significantly increased secretion of VEGF (p≤0.001) and TGF-β1 (p=0.028) by pericytes under hypoxic culture conditions was detected by ELISA while secretion of Ang-1 was reduced by 35% (p>0.05). Very little secretion of Ang-2 was detected under both conditions (p>0.05). (D) Immunohistochemistry revealed human VEGF₁₆₅ expression by GFP-labeled donor pericytes within the infarct area at 2 weeks post-infarction (a) merge (b) hVEGF₁₆₅ in red (c) anti-GFP in green (scale bar=50μm).

Figure 7. Transplanted pericytes home to perivascular locations

(A) Pericytes were transduced with GFP reporter at nearly 95% efficiency. Fluorescence ([A], main) and bright-field ([A], inset) images were taken from the same low-power field (scale bars=200μm). (B) Engraftment of GFP-labeled pericytes within host myocardium was revealed by anti-GFP immunostaining at 1 week post-injection (scale bar=500μm, infarct site encircled by dotted lines). (C) Pericytes were lining with ([C], main) or surrounding ([C], inset) host CD31-positive microvasculature (scale bars=20μm). (D) The engraftment efficiency of pericytes at 1 week (9.1±1.3%) and 8 weeks (3.4±0.5%) post-infarction was depicted (dash-dot line). The perivascular homing ratio instead increased from 28.6% to 40.1% and was delineated separately (solid line). Some GFP-positive pericytes juxtaposing host ECs (E) expressed human-specific EphB2 (green/white arrows) or (F) formed connexin43-positive gap junctions with ECs (red arrow heads) (scale bars=10μm).